U.S. patent number 4,208,398 [Application Number 05/570,254] was granted by the patent office on 1980-06-17 for technetium-labeled complexes, production and use thereof.
This patent grant is currently assigned to Hoffman-La Roche Inc.. Invention is credited to Theodore F. Bolles, David O. Kubiatowicz.
United States Patent |
4,208,398 |
Kubiatowicz , et
al. |
June 17, 1980 |
**Please see images for:
( Certificate of Correction ) ** |
Technetium-labeled complexes, production and use thereof
Abstract
Novel chemical complexes containing a radioactive technetium
isotope are kidney specific when the complexing agents are certain
ionic, water-soluble mercaptans. The complexes can be made by
reducing pertechnetate ion and reacting the reduced technetium
species with the mercaptan. The complexes are normally used in a
biologically sterile, substantially isotonic aqueous medium, for
diagnostic purposes.
Inventors: |
Kubiatowicz; David O. (Arden
Hills, MN), Bolles; Theodore F. (Woodbury, MN) |
Assignee: |
Hoffman-La Roche Inc. (Nutley,
NJ)
|
Family
ID: |
26997884 |
Appl.
No.: |
05/570,254 |
Filed: |
April 21, 1975 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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353298 |
Apr 23, 1973 |
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Current U.S.
Class: |
424/1.65;
424/1.73; 534/14 |
Current CPC
Class: |
A61K
51/0402 (20130101); A61K 51/0491 (20130101); A61K
2123/00 (20130101) |
Current International
Class: |
A61K
51/02 (20060101); A61K 51/04 (20060101); A61K
029/00 (); A61K 043/00 () |
Field of
Search: |
;424/9 |
References Cited
[Referenced By]
U.S. Patent Documents
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3873680 |
March 1975 |
Jackson et al. |
3928552 |
December 1975 |
Winchell et al. |
|
Primary Examiner: Padgett; Benjamin R.
Assistant Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Saxe; Jon S. Leon; Bernard S.
Epstein; William H.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of our co-pending
application Ser. No. 353,298, filed Apr. 23, 1973, abandoned.
Claims
What is claimed is:
1. A complex of Tc-99m with a compound of the group consisting of
dimercaptosuccinic acid, mercaptosuccinic acid, mercaptoacetic
acid, mercaptopropionic acid, 3-mercaptopropylsulfonic acid,
thioglucose or thiolactic acid.
2. A complex according to claim 1, in which the complex contains
from about 1 to 100 millicuries of Tc-99m for each milligram of
complexing agent.
3. A composition comprising an isotonic solution of complexing
agent according to claim 1 in a pharmaceutically acceptable
parenteral diluent.
4. Composition according to claim 3, in which the parenteral
diluent is sterile aqueous isotonic saline solution.
5. A complex according to claim 1, in which the complexing agent is
dimercaptosuccinic acid.
6. A complex according to claim 1, in which the complexing agent is
thioglucose.
7. A complex according to claim 1, in which the complexing agent is
mercaptoacetic acid.
8. A complex according to claim 1, in which the complexing agent is
3-mercaptopropanoic acid.
9. A radiopharmaceutical complex of technetium-99m with a
complexing agent selected from the group consisting of
mercaptoacetic acid, dimercaptosuccinic acid, mercaptosuccinic acid
and mercaptopropionic acid, said complex prepared in the presence
of stannous chloride reducing agent.
10. The radiopharmaceutical complex of claim 9 wherein said agent
is dimercaptosuccinic acid.
11. A radiopharmaceutical complex of technetium-99m with a
complexing agent selected from the group consisting of
mercaptoacetic acid, dimercaptosuccinic acid, mercaptosuccinic acid
and mercaptopropionic acid, said complex prepared in the presence
of a pharmaceutically-acceptable reducing agent for
pertechnetate.
12. A radiopharmaceutical complex of reduced technetium-99m with a
complexing agent selected from the group consisting of
mercaptoacetic acid, dimercaptosuccinic acid and mercaptopropionic
acid, wherein the technetium-99m is reduced by stannous
chloride.
13. The radiopharmaceutical complex of claim 12 wherein said agent
is dimercaptosuccinic acid.
Description
FIELD OF THE INVENTION
This invention relates to chemical complexes of the radioactive,
metastable isotope technetium-99m (Tc-99m) wherein the complexing
agents are selected from certain water-soluble mercaptans. A
further aspect of this invention relates to a process for producing
the chemical complex and a preferred biologically sterile,
substantially isotonic medium containing the complex. Still another
aspect of this invention relates to the use of the products of the
invention in studies of kidney structure and function.
DESCRIPTION OF THE PRIOR ART
The art of radiochemistry has found many applications in the fields
of medicine and biology. It has long been known that the
introduction into an organism of compounds containing (or "labeled"
with) a radioisotope can provide insight into the anatomy and
physiology of the organism. These compounds, generally referred to
as radiopharmaceuticals, are particularly useful in diagnostic
techniques which involve studying the structure or function of
various internal organs, e.g. the kidney, with radiation detection
means. For diagnostic work, isotopes with a short half-life and an
emission spectrum rich in gamma rays (as opposed to beta particles)
are preferred.
The metastable isotope Tc-99m has a six hour half-life and an
emission spectrum, 99 percent gamma radiation at 140 KeV, which is
extremely well-suited for techniques of diagnostic nuclear
medicine. Thus, Tc-99m has a high specific activity,
5.28.times.10.sup.9 millicuries per gram (mCi/g), and a
conveniently rapid rate of decay; whereas its daughter product,
Tc-99, has a specific activity which is almost nine orders of
magnitude lower and a half-life which is roughly eight orders of
magnitude longer. For the organism being studied or diagnosed, the
slow rate of decay from the relatively stable, lower specific
activity Tc-99 to its degradation product (ruthenium) would not
normally produce any hazardous amounts of radiation, regardless of
the biological means or route of elimination of a Tc-99m
radiopharmaceutical. For the researcher or clinician, the emission
spectrum of Tc-99m can provide high levels of accuracy in
radiodiagnostic measurements and calculations. In recent years,
Tc-99m has become readily available in hospitals through the use of
selective elution from a so-called molybdenum-99 (Mo-99) generator.
The isotope Mo-99 produces Tc-99m as a radioactive decay
product.
Although Tc-99m compounds would appear to be ideal
radiopharmaceuticals for diagnostic use, providing or selecting Tc
compounds or complexes with a view toward organ specificity and
tolerable levels of toxicity is not a simple matter of selection.
Obviously, compounds with a very high toxicity are undesirable for
human or veterinary use, even in the small amounts called for by
diagnostic work. Compounds with insufficient in vivo stability may
be poor diagnostic tools, since radioactive ions or other chemical
species with insufficient or undesired organ specificity may be
liberated. Stable compounds which become distributed generally
throughout the organism, despite their stability, or which do not
reach a desired destination in the organism are also poorly suited
for many studies of organ function or structure, e.g. kidney
studies.
The problem of selecting or preparing a kidney specific
radiopharmaceutical for kidney imaging or function studies is
particularly difficult. Both the liver and the kidney are capable
of removing various types of compounds from the body--ultimately
through excretion in feces and urine, respectively. Any
radiopharmaceutical used for kidney studies should ideally have
maximal kidney specificity and minimal liver specificity. A number
of biological and chemical factors must be considered and brought
under control before the desired organ specificity and route of
excretion can be obtained.
For the study of kidney structure, for example, Tc-99m-containing
agents are preferably more slowly cleared, or even quantitatively
absorbed and retrieved by kidney tissue, so as to permit
visualization of kidney structure by scanning instruments. An
example of a useful agent of this type is .sup.99m Tc-dimercapto
succinic acid. Under certain circumstances, however, as where
modern high-speed gamma cameras are available, kidney structure can
be studied using technetium compounds which are rapidly cleared
from kidney tissue such as thiosaccharide .sup.99m Tc-thioglucose
and .sup.99m Tc-mercapto succinic acid as disclosed herein.
Technetium-99m compounds have been used in brain or other organ
scanning. For example, Tc-sulfur colloid can be used for liver
scanning.
Representative of the literature relating to the radiopharmacology
of Tc-99m compounds are the following articles:
Larson et al, J. Nuclear Medicine, 7:8:7 (1966) relating to
Tc-99m-colloid preparation for photo-scanning, lung imaging and
pancreas imaging;
Tubis et al, International Journal of Applied Radiation and
Isotopes, V. 19, 835 (1968), relating to Tc-99m-labeled cystine,
methionine, and a synthetic polypeptide and their distribution in
mice; and
U.S. Pat. No. 3,466,361, showing preparation of chelates of
technetium and their use for various diagnostic purposes.
Compared to the common transition metals, very little is known
about the chemistry of technetium. Technetium belongs to Group
VII-B of the Periodic Table; its chemistry bears a superficial
resemblance to manganese but tends to be more similar to the higher
member of the Group, rhenium. Technetium can apparently exist in a
range of oxidation states, including +7 (e.g. pertechnetate) and
several lower oxidation states, some of which are difficult to
characterize and/or are relatively unstable. In spectrophotometric
determinations of technetium, the element has been complexed with
toluene-3,4-dithiol, thioglycolic acid and thiocyanates. See Miller
et al, Anal. Chem., page 404 (1961) and page 1429 (Oct. 1960), and
Crouthamel, Anal. Chem., page 1756 (Dec. 1957).
The complexes of the present invention must not, however, be
confused with the colored complexes used for analysis as described
in these references. The analytical procedures use colorimetric
techniques. It seems clear that these are formed using far higher
concentrations of technetium than those used in the present
invention; and it is believed that the colored substances and
solutions used for analytical purposes for determination of
technetium do not suggest the present pharmaceutically acceptable
nor their unique biological behavior or use for the diagnostic
methods described herein.
Accordingly, this invention contemplates providing pharmaceutically
acceptable complexes of Tc-99m which have sufficient in vivo
stability and sufficiently low toxicity for use in humans or
animals and which preferably are:
Removed from the blood or other vital organs or tissues by the
kidney rather than by, for example, the liver or the lungs;
Concentrated in the kidney at a high rate if visualization is
desired;
Concentrated in other organs or tissues--particularly organs or
tissues in close proximity to the kidney--at a very low or
negligible rate;
Eliminated from the body by alternative routes to a minor,
preferably negligible, extent.
This invention further contemplates means and methods whereby Tc
complexes can be most efficiently produced and utilized for kidney
structure studies.
BRIEF SUMMARY OF THE INVENTION
Briefly, this invention involves reducing an appropriate amount of
radioactive pertechnetate ion (.sup.99m TcO.sub.4.sup.-) until a
major amount of the pertechnetate ion has been reduced to a
technetium species having an oxidation state greater than 0 but
less than +7 and then reacting this technetium species with an
excess of one of the subsequently described sulfur-containing
complexing agents. The resulting Tc-99m complex is suitable for
injection into the blood stream of a mammal when dissolved or
dispersed in a biologically sterile, aqueous medium substantially
isotonic with mammalian body fluids. The reduction step can be
carried out chemically through acid catalysts if the complexing
agent is also a reducing agent, at least when the complexing agent
is present in large excess, as will normally be the case.
Preferably, however, reduction is achieved through an iron (II)
salt, a copper (I)/copper (II) couple, a tin (II) salt, or a
combination of two or more of these agents.
A meaningful evaluation of kidney function can be obtained by
measuring the increase in and loss of radioactivity from the kidney
of the animal or patient being studied. It will generally not be
necessary to monitor the radioactivity for more than about 24 hours
after the injection, and 12 hours of monitoring can be fully
sufficient.
Kidney structure can be determined by gamma radiation detectors to
measure and record the radioactivity emitted from the kidney.
The complexing agents useful for the purpose of the invention are
thiol-group-containing, pharmaceutically acceptable, water-soluble
organic acids having from 2 to 15 carbon atoms. In addition to the
thiol group they contain as acidic moieties hydroxyl, carboxylic or
sulfonic groups.
They also exhibit certain oil-water partition behavior.
DETAILED DESCRIPTION OF THE INVENTION
The complexing agents of the invention are organic thiols which are
capable of providing a kidney-specific, radioactive
technetium-containing compound suitable for inclusion in injectable
media substantially isotonic with mammalian body fluids. These
compounds are rapidly removed from the blood or other tissues by
the kidney, are present in the kidney for a time and then excreted
by the kidney into the bladder, and therefore removed from the body
more or less completely by way of the urine. Thus, the selection of
complexing agents according to this invention involves weighing a
combination of chemical and biological criteria. Chemical
consideration of the complexing agent alone does not insure that
the technetium complex will have a kidney specificity,
compatibility with blood or other mammalian body fluids, or the
like.
The compounds useful as complexing agents for technetium, which are
to be used as diagnostic agents for kidney structure and function
are water-soluble and stable at the pH of the blood, namely about
pH 7.4. In every case they contain a mercaptol or thiol, i.e. -SH
group, which is the site of complexing with the Tc-99m.
Broadly speaking, the complexing agents of the invention are
aliphatic thiol compounds and certain thiosaccharides having from 2
to 15 carbon atoms, to which are attached, as substituents, one or
two thiol groups, together with from one to five acidic functional
groups of the class consisting of hydroxyl, carboxylic or sulfonic
groups, not more than three carboxylic groups or one sulfonic group
being present. In every case there is at least one of the described
functional groups present for every three carbon atoms in the
compound. The aliphatic carbon chains which are present are
straight or branched.
The complexes of the invention are soluble to the extent of about
0.01 percent to 100 percent by weight in water. A useful test to
determine whether the complexes are useful for the purposes of the
invention is the determination of the partition coefficient of the
complex between water and octanol (hydrophilicity). This ratio of
distribution is quite easily followed by determining the activity
of the respective phases of a two-phase system, in which the
complex is added as a water solution to octanol in equal parts. In
this system, complexes useful for the purposes of the invention
show the following relationship ##EQU1## (wherein a.sub.w is the
radioactivity distributed in the aqueous phase and a.sub.o is the
radioactivity distributed in the organic phase, at a pH in the
range between pH 4 and pH 9), after thorough agitation to admix the
phases followed by standing to accomplish complete separation.
Further, the partition coefficient is relatively uniform throughout
the stated pH range; i.e., does not change more than about three
units over this range.
The following table illustrates the water/octanol partition
coefficient for Tc-99m-dimercaptosuccinic acid in a concentration
of 0.027 mole/liter:
TABLE I ______________________________________ pH Partition
Coefficient = Ln(a.sub.w /a.sub.o)
______________________________________ 4 6.0 5 6.8 6 7.6 7 8.5 8
8.2 9 8.1 ______________________________________
In addition, the Tc-99m complexes of the invention, when
chromatographed on unactivated, 100-micrometer thick silica gel
sheet with polyvinyl alcohol binder at neutral pH, and developed
with anhydrous acetone, are not desorbed, i.e. show Rf=0, whereas
unreacted aqueous pertechnetate shows Rf=1.0. However, when
developed with anhydrous acetone:concentrated HCl in a
volume/volume ratio of 100:0.5, the complexes of the invention show
Rf=about 0.6.
Typical of the compounds useful for forming the complexes with
Tc-99m are dimercaptosuccinic acid, dimercapto glutaric acid,
dimercapto adipic acid, mercaptosuccinic acid, mercaptoacetic acid,
mercaptopropionic acid, 3-mercaptopropylsulfonic acid, thioglucose
and thiolactic acid.
The term "substantially isotonic with mammalian body fluids", as
used herein, denotes the condition when the osmotic pressure
exerted by the solution in question is sufficiently similar, as
compared to a body fluid such as blood, so that no dangerous hypo-
or hypertonic condition results in the patient or test animal when
0.1 ml. (in the case of a mouse) or up to 10 ml. (in the case of a
human) of the solution is injected into the patient's or animal's
bloodstream.
The exact mechanism by which the complexing agents used in this
invention become chemically linked to technetium is difficult to
determine. It appears that the Tc-99m should be present primarily
in an oxidation state of at least about +3 but not more than +6.
This oxidation state can be conveniently obtained by reducing
Tc-99m-pertechnetate, a relatively stable +7 technetium species.
The reduced species can coordinate with one or more sulfur atoms
which are in the form of mercaptan groups.
The amount of Tc-99m needed to produce an amount of
radiopharmaceutical suitable for most diagnostic or research uses
is extremely small and is generally in the range of about 0.01
millicurie per milliliter (mCi/ml) of 99m-pertechnetate solution up
to about 500 mCi per ml. of such solution. Only about
0.02.times.10.sup.-10 gram of 99m-pertechnetate dissolved in a
milliliter of aqueous medium (e.g. isotonic saline) is needed to
provide 0.01 mCi/ml, and less than 100.times.10.sup.-10 gram of
99m-pertechnetate per milliliter of solution provides enough
radioactivity for most uses.
Owing to the short half-life of the Tc-99m, it is preferred to
prepare small batches of 99m-pertechnetate solution for immediate
use. Batches as small as 0.1 ml. can be adequate for animal studies
(e.g. for injection in mice), and batches as large as 50 ml. are
convenient for one or more injections in one or a group of human
patients. In any event, it would be a rare situation that required
more than about 100.times.10.sup.-10 gram (i.e. about 10.sup.-10
gram-atoms) of Tc-99m as pertechnetate ion to produce a few ml. of
radiopharmaceutical, regardless of stoichiometry of the Tc complex.
It is preferred to provide enough complexing agent (ordinarily at
least 5.times.10.sup.-9 moles per milliliter of reaction mixture)
to have an excess over stoichiometry with respect to the Tc-99m in
the reaction mixture. A large excess of complexing agent (e.g.
0.5-1000 micromoles of complexing agent per ml. of reaction
mixture) can be desirable, particularly when the complexing agent
itself serves as the means for reducing the oxidation state of
pertechnetate.
The Tc-99m used in this invention is obtainable from a Mo-99
generator in the conventional manner. Eluting or "milking" the
generator with an aqueous medium will provide the
Tc-99m-pertechnetate solution in the form of M.sup.+x
(99m-TcO.sub.4.sup.-).sub.x, where M.sup.+x is a pharmaceutically
acceptable cation such as a proton, an alkali metal ion, an
ammonium ion or the like, and x is a positive integer less than
four. Typically, the aqueous elution medium is a saline solution,
which provides sodium 99m-pertechnetate.
The pertechnetate ion can be reduced chemically or electrolytically
to a lower oxidation state of technetium, preferably by reaction
with an oxidizable low valence metal salt such as a tin (II) salt
(e.g. SnCl.sub.2), an iron (II) salt (e.g. a ferrous salt/ascorbic
acid medium), a Cu(I)/Cu(II) couple, a combination thereof, or
other chemical reducing agents such as mercaptans, metal hydrides,
thiosulfates, hypophosphites, bromides, iodides, etc.
To avoid possible accidental contamination of the complex with
undesirable metal ions, the preparation steps are carried out in
glass vessels, and the use of hypodermic needles for transferring
solutions of the mercaptan compounds is avoided.
A particularly suitable means for providing the reducing agent and
complexing agent is to pre-formulate a radiopharmaceutical kit for
use with the No-99 generator. For example, 0.1 (preferably at least
0.5) to 10 ml. of a solution containing about 0.5 to about 1000
micromole/ml. of complexing agent and a suitable amount, e.g.
0.01-100 micromole ml. of reducing agent can be hermetically and
aseptically sealed in separate vials or the same vial. The contents
of the vial or vials can further be treated (for example by freeze
drying) to produce a dry powder. A preservative such as benzyl
alcohol is optionally included in the contents of the vial. The
solution in the vial or an aqueous solution of the dry powder is
preferably substantially isotonic with mammalian body fluids, e.g.
human blood. The contents of the vial can be combined with the
pertechnetate-containing, substantially isotonic eluate, mild heat
can be applied if necessary to the combined solutions to achieve
the reduction and Tc-complex formation, and the resulting
radiopharmaceutical can then be injected into the blood of the
patient or test animal.
Conveniently, the container is provided with a plunger means and
means for attaching a hypodermic needle so that the vial functions
as a hypodermic syringe, whereby the contents after preparation of
the solution of Tc-99m complex can be injected parenterally without
being transferred to another container or syringe.
Radioactivity measurements are made in the conventional manner for
a period from the time of injection until about 24 hours
afterwards, depending on the nature of the study or diagnosis. Most
studies call for at least one-half hour of post-injection
radioactive measurements. These measurements can be corrected for
decay in the usual manner and studied with a view toward obtaining
a picture of kidney structure or a measure of kidney function.
The amount of complexing agent injected into a test animal or human
patient should preferably be less than 25 percent (e.g. less than
10 percent) of the LD 50 in mg. per kg. of body weight, though
higher amounts are permissible in veterinary medicine. Typical LD
50's (determined in rodents and at least one other species) for
preferred complexing agents of this invention range from about 20
to 1000 mg. per kg. of body weight.
When optical isomerism is possible, as in the case of
dimercaptosuccinic acid, DL-racemic mixtures are fully operative in
the invention and are easier to synthesize than the individual
isomers. If desired, however, racemic mixtures can be resolved by
conventional techniques.
Acid, salt or hydroxyl groups present on the complexing agent
molecule can provide a water-solubilizing or hydrophilic effect
which is reflected in higher L.sub.n (a.sub.w /a.sub.o) values, but
due regard must be accorded to the variety of fluids, organs and
tissues in mammals, each of which can have a distinctively acidic
or basic environment, ranging from, for example, the low pH of the
stomach to the relatively high pH of the intestines. The blood is
on the mildly alkaline side at pH=7.4, while the urine, etc., is
about pH 5 to pH 8. Thus, partition coefficient data on the
Tc-complexes of this invention are preferably obtained throughout
the pH range of 4 to 9. The use of partition coefficient data in
pharmacology is well-established; see Andrejus Korolkvas,
Essentials of Molecular Pharmacology, Wiley (Interscience), New
York (1970). It has now been found that the water/n-octanol system
provides useful data for evaluating lipophilic-hydrophilic balance
of Tc-complexes without in vivo testing. Natural logarithms of
partition coefficients are tabulated in several of the examples
which follow.
Due regard should also be given to chelating effects of the
water-solubilizing groups COOH, SO.sub.3 H and OH.
In the preparation of the complexes, when the complexing agents of
this invention are used together with an oxidizable lower valence
metal salt, the salt can be combined with a water solution of the
complexing agent. For example, mercaptosuccinic acid can be
dissolved in a sodium bicarbonate-water solution, and a reducing
agent comprising an excess over stoichiometry of
dissolved in ethanol or 1 molar aqueous HCl can then be added to
the solution. After the complexing and reducing agents have been
combined, 99m sodium pertechnetate can be added. Agitation at a
normal ambient temperature (20.degree.-25.degree. C.) will initiate
the reduction step, and over 50 percent (in practice more than 80
percent) of the pertechnetate ion will be in reduced form after
less than an hour at this ambient temperature. The extent of
reduction can be determined with thin layer chromatography (T.L.C.)
and radiation monitoring, since TcO.sub.4.sup.- and its
reduced-and-complexed form have distinctly different R.sub.f values
if the chromatogram is developed with properly selected
solvents.
If the oxidizable low valence metal salt is omitted, the sodium
pertechnetate eluate can be reacted with HBr to form
H.sub.2.sup.99m TcBr.sub.6. This reaction is preferably carried out
by repeatedly evaporating the eluate in the presence of 0.1 N (or
more concentrated) aqueous HBr under an atmosphere of dry, inert
gas such as nitrogen. The H.sub.2 TcBr.sub.6 can be extracted with
acetone, reacted with an excess of the mercaptan complexing agent
in a non-aqueous medium to form the Tc complex, and then worked up
in saline solution or the like. Further pH changes can be used, if
necessary, to dissolve the Tc complex.
The amount of radioactive .sup.99m Tc required for use in imaging
is relatively very small, being of the order of 1 to 100
millicuries of .sup.99m Tc per mg. of complexing agent. A total
amount of 2 millicuries of radioactive material typically suffices
for test purposes in the average (70 kg.) human.
It is much more convenient to measure out larger amounts of
complexing agent than that which is just sufficient to complex with
the very small amount of radioactive material. Any residual acidic
function of the complexing agent is conveniently neutralized using
a solution of sodium hydroxide or sodium bicarbonate. The tonicity
of the agent is adjusted to substantially isotonic with sodium
chloride, if necessary. The substantially isotonic
radiopharmaceutical is then ready for injection.
The distinct R.sub.f values of novel Tc-mercaptan compounds or
complexes produced according to this invention can reliably
characterize these compounds so that they are distinguished from
their precursors. Since only minute amounts of complexes of Tc-99m
are produced, analysis of the complex by any method other than
T.L.C. is extremely difficult at best. To reproducibly determine
the R.sub.f values, thin layer chromatographs can be made from
appropriate solutions and a standardized chromatogram sheet.
Reproducible results have been obtained with unactivated, 100
micron thick silica gel chromatogram sheets having a polyvinyl
alcohol binder and a neutral pH. One commercially available form of
such a chromatogram is designated Eastman Chromagram Sheet
6060.
EXAMPLE 1
Preparation of the Kidney Specific, Technetium Complex with
Dimercaptosuccinic Acid
About 5 millicuries of 99m-TcO.sub.4 (10.sup.-9 gms.) in 2 ml. of
saline solution (as an eluate from a Mo-99-Tc-99m generator) were
placed in a 10 ml. glass vial. Two ml. of 48 percent aqueous HBr
were added to the vial, and the vial contents were evaporated to
dryness over a steam bath under a N.sub.2 atmosphere. This
evaporation procedure was repeated twice, resulting in the
formation of TcBr.sub.6.sup.-2.
Ten milligrams of dimercaptosuccinic acid (DMSA) in about 3 ml. of
absolute ethanol were added to the TcBr.sub.6.sup.-2 residue in the
vial. The alcoholic solution was evaporated to about 0.5 ml.
volume.
Next, 9 ml. of saline were added to the glass vial (held under
N.sub.2 atmosphere). The pH of the vial contents was raised to pH
7.4, using 1 molar NaOH. The solution was transferred to a
pharmaceutical vial, capped and sealed. Using a needle inserted
through the rubber seal septum, the vial was evacuated to 5 mm. Hg
pressure and purged with nitrogen to 1 atmospheric pressure. The
vial contents were ready for injection and kidney imaging. Good
kidney images in dogs were obtained one-half hour after injection
of this solution, using a gamma radiation detecting camera.
Table II shows the distribution of 99mTc-DMSA in white female Swiss
Webster mice, each weighing about 20 grams. The mice were injected
intravenously via the tail vein, sacrificed after appropriate time
periods, then dissected. The isolated organs were assayed for
radioactivity using a Packard series 410 A Auto Gamma Spectrometer.
Percent activity distribution was calculated (allowance was made
for the radioactive decay of Tc-99m) and is shown in the table.
TABLE II ______________________________________ Biological
distribution of 99m-Tc-dimercaptosuccinic acid in mice as a
function of time Percentage of total 99m-Tc in mice as a function
of time Organ 0 hr. 0.5 hr. 1.0 hr. 2.0 hr. 4.0 hr. 24.0 hr.
______________________________________ Lungs 7.4 2.3 1.7 0.8 0.7
0.0 Liver 28.6 12.8 9.4 6.8 3.8 2.7 Spleen 0.3 0.4 0.2 0.3 0.1 0.1
Kidneys 9.4 29.3 39.5 45.6 54.8 75.5 Stomach 1.8 1.1 1.2 0.5 0.4
0.4 Gut 10.1 6.1 7.0 4.3 3.0 1.7 Pancreas 1.4 0.7 0.4 0.3 0.2 0.2
Carcass 40.9 30.4 26.9 20.4 21.3 8.0
______________________________________
Each data point represents an average value from three mice taken
in three separate distribution studies.
Activity begins to accumulate in the animal's kidneys immediately
after injection of the Tc-DMSA complex and continues to concentrate
during a 24 hour period. These data can be compared to mouse kidney
distribution data of Tc-99m pertechnetate, which concentrates to a
maximum of 3 percent at any time during a 24 hour period.
Thin layer chromatographic analysis of the Tc-DMSA complex was
performed as follows:
Chromatogram: Unactivated 100-micrometer thick silica gel sheet
with polyvinyl alcohol binder, neutral pH (Eastman Chromagram
6060).
Solvent Systems: (1) anhydrous acetone; (2) anhydrous
acetone-concentrated aqueous HCl (36 percent) in a volume/volume
ratio of 100:5.
Developed Chromatograms: Tc-DMSA developed with (1), Rf=0, whereas
unreacted pertechnetate had an Rf=1.0. Tc-DMSA developed with (2),
Rf=0.63, whereas unreacted pertechnetate had an Rf=1.0.
EXAMPLE 2
Preparation of Technetium Dimercaptosuccinic Acid for Kidney
Imaging using Mercaptan Reduction in HCl.
Twenty-five mg. of dimercaptosuccinic acid were added to a 20 ml.
pharmaceutical vial containing 1 millicurie of 99m-TcO.sub.4.sup.-
in 4 milliliters of 0.5 molar HCl. The vial was closed, vented with
a hypodermic needle, and placed in a boiling water bath for 10
minutes.
One molar NaOH was added to the cooled vial until the pH of the
solution was raised to 7.6. The vial was evacuated and purged with
N.sub.2. The solution of dimercaptosuccinic acid complex was then
ready for injection.
EXAMPLE 3
Preparation of Kidney Specific Agent Technetium-thioglucose
About 1 millicurie of 99m-TcO.sub.4.sup.- in 0.2 milliliter of
saline solution was added to a glass vial. Two milliliters of 48
percent HBr were added to the vial, and the vial contents were
evaporated to dryness over a steam bath under a N.sub.2 atmosphere.
The evaporation procedure was repeated twice.
The residue of 99m-TcBr.sub.6.sup.- was dissolved by adding two
milliliters of ethanol. Twenty-five milligrams of sodium
thioglucose were added, and the solution was evaporated to near
dryness using a stream of warm N.sub.2.
Ten milliliters of saline solution were added to dissolve the
99m-Tc-thioglucose complex. The solution pH was 7.4.
Thin layer chromatographic analysis of the Tc-thio-glucose complex
was performed as described in Example 1. The results were:
Tc-thioglucose developed with (1), Rf=0. Unreacted pertechnetate
had an Rf=1.0. Tc-thioglucose developed with (2), Rf=0.63 and
0.75.
Table III shows the distribution of 99m-Tc-thioglucose in female
white Swiss Webster mice. The mice were tested and assayed for
radioactivity as described in Example 1.
TABLE III ______________________________________ Biological
distribution of 99m-Tc-thioglucose in mice as a function of time.
Percentage of total 99m-Tc in mice as a function of time. Organ 0
hr. 0.5 hr. 1 hr. ______________________________________ Lungs 2.9
0.3 0.08 Liver 18.9 2.1 3.3 Spleen & Pancreas 2.1 0.1 0.2
Kidneys 9.0 2.8 0.4 Stomach 0.8 0.8 0.06 Gut 13.3 5.5 1.1 Carcass
53.1 1.5 1.4 Urine 0.0 89.9 93.5
______________________________________
This compound is eliminated from the kidneys of the mice very
rapidly as shown by the percentages of technetium activity found in
the various organs during the test time periods. The "0 hr." test
data do not indicate compound specificity but rather blood flow to
various organs.
Technetium-thioglucose can be used for kidney imaging when a gamma
camera is employed, or it can provide a measure of kidney function
by monitoring its disappearance from the animal's bloodstream.
EXAMPLE 4
Preparation of Kidney Specific Agent Technetium-mercaptoacetic
Acid
About 3 millicuries of 99m-TcO.sub.4.sup.- were added to a glass
vial. One milliliter of 48 percent HBr was added to the vial, and
the vial contents were evaporated to dryness over a steam bath
under a N.sub.2 atmosphere. The evaporation procedure was repeated
twice.
Twelve milligrams of mercaptoacetic acid dissolved in 4 milliliters
of acetone were added to the dry 99m-TcBr.sub.6.sup..dbd. residue.
After 20 minutes the acetone solution was evaporated to near
dryness, then dissolved with 5 milliliters of saline solution. 0.5
Percent of sodium hydroxide solution was added to adjust the
solution pH to 7.4.
One hour after this complex was injected into the blood stream of a
mouse, about 24 percent of technetium activity had localized in its
kidneys. A large part of the remaining activity was found in the
animal's urine.
The partition coefficient Ln(a.sub.w /a.sub.o) for
technetium-mercaptoacetic acid is shown in the following table:
TABLE IV ______________________________________ Solution pH
Partition Coefficient - Ln(a.sub.w /a.sub.o)
______________________________________ 4 2.9 5 3.2 6 3.5 7 3.8 8
4.1 9 4.4 ______________________________________
EXAMPLE 5
Preparation of Kidney Specific Technetium-3-mercaptopropanoic
Acid
About 12 milligrams of 3-mercaptopropanoic acid were added to an
acetone solution of 99m-TcBr.sub.6.sup..dbd. (prepared as in
Example 4). After 20 minutes reaction time the acetone solution was
evaporated to near dryness, diluted to 5 milliliters volume with
saline and adjusted to pH 7.4 with 0.5 percent sodium hydroxide.
The solution was ready for kidney imaging studies.
The partition coefficient of this technetium complex is shown in
the following table:
TABLE V ______________________________________ Solution pH
Partition Coefficient - Ln(a.sub.w /a.sub.o)
______________________________________ 4 2.4 5 2.7 6 3.1 7 3.6 8
4.1 9 4.7 ______________________________________
EXAMPLE 6
Preparation of Technetium-mercaptosuccinic Acid Complex using Cu II
as Catalyst
A solution was prepared containing 50 milligrams of
mercaptosuccinic acid in 1.0 ml. of saline, in a 20 ml.
pharmaceutical vial. The pH of the solution was approximately
2.
A solution of 30 micrograms of CuSO.sub.4 5H.sub.2 O in 10
microliters of saline was added to the vial.
The vial was stoppered, capped and purged with N.sub.2.
To prepare the technetium complex of mercaptosuccinic acid, 0.4
millicurie of 99m-TcO.sub.4 from a Mo-99-Tc-99m generator was added
to the vial in 5 milliliters of saline.
The solution was heated for at least five minutes in a boiling
water bath to complete the reaction. Afterwards the solution was
neutralized to 7.0-7.4 pH, using 1 molar NaOH solution.
FeCl.sub.3 can be used instead of the CuSO.sub.4 in this
preparation.
Reference has been made herein to thiosaccharides, and these
compounds are commonly described in the art as having cyclic
structure. They can also exist in tautomeric form as open-chain
compounds, and for the purpose of this invention they are to be
included within the designation "aliphatic".
* * * * *